Reagent for semi-quantitative one-pot real-time nucleic acid detection and detection method

Abstract

The invention relates to the technical field of biology, in particular to a reagent and a detection method for real-time nucleic acid detection by a semi-quantitative one-pot method. Aiming at a one-pot method recombinase polymerase amplification (RPA) and CRISPR detection (CRISPR-Dx) reaction, a series of one-pot method RPA-CRISPR-Dx systems with different detection limits are constructed by redesigning an RPA primer, screening a CRISPR target PAM and combining the RPA primer and the CRISPR target PAM. A detection array constructed on the basis of the detection systems can output semi-quantitative results which do not need to be calculated and processed and can be read directly, and a powerful tool is supplemented for improving the real-time nucleic acid detection quantitative capacity.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to reagents and detection methods for semi-quantitative one-pot instantaneous nucleic acid detection. Background Technology

[0002] Nucleic acid testing, as a fundamental technology in molecular biology, is widely used in disease diagnosis, infectious disease surveillance, environmental monitoring, and food safety. To effectively support clinical decision-making and public health management, nucleic acid testing methods not only need high sensitivity and accuracy but also require rapid testing, simple operation, and low equipment dependence. However, while the widely adopted real-time quantitative polymerase chain reaction (qPCR) technology demonstrates excellent analytical performance, it relies on a sophisticated thermal cycling control system and real-time optical signal acquisition device. This results in large equipment size, high cost, and a relatively complex testing process, hindering its widespread application in point-of-care (POC) testing and resource-constrained environments. Therefore, improving the accessibility and applicability of testing methods while ensuring testing performance has become a crucial direction for the development of nucleic acid testing technology.

[0003] To reduce reliance on complex instruments, various nucleic acid detection technologies based on isothermal reaction conditions have been developed. Among them, nucleic acid detection methods based on the CRISPR-Cas system have attracted widespread attention due to their highly programmable sequence recognition capabilities and excellent specificity. Guided by guide RNA, Cas nucleases can specifically recognize and cleave target nucleic acid sequences. Some Cas nucleases also exhibit non-specific paracleavage activity after target recognition, which can be used to amplify the detection signal. By combining CRISPR nucleic acid detection with isothermal nucleic acid amplification technology, detection sensitivity can be further improved, bringing its analytical performance close to qPCR levels. To simplify the operation process and reduce the risk of aerosol contamination, related technologies have gradually developed into one-pot detection modes that integrate nucleic acid amplification reactions and CRISPR-mediated detection into the same reaction system.

[0004] However, in one-pot detection systems, nucleic acid amplification and Cas-mediated cleavage reactions occur simultaneously in the same reaction environment, resulting in a complex kinetic coupling between the two. When the Cas-mediated cleavage rate is high, the target nucleic acid may be consumed before it is fully amplified, thus affecting detection sensitivity; conversely, a low cleavage rate may delay signal generation or increase background interference. Therefore, the dynamic balance between the amplification and CRISPR cleavage reactions becomes a key factor affecting the performance of one-pot detection. To address these issues, existing technologies typically improve detection performance by optimizing the parameters of a single detection reaction system. However, these methods often rely on maintaining reaction equilibrium within a narrow parameter window, making them sensitive to fluctuations in reaction conditions and difficult to balance sensitivity, stability, and repeatability. Furthermore, because target amplification and cleavage reactions occur simultaneously in one-pot detection, the generation of the detection signal is influenced by multiple factors, making it difficult to establish a stable and reproducible quantitative relationship between the signal output and the initial concentration of the target nucleic acid. Therefore, existing one-pot CRISPR nucleic acid detection methods are mostly used for qualitative analysis. While existing research has attempted to achieve quantitative detection through signal fitting, reaction time analysis, or digital microreaction units, these approaches typically rely on complex data processing algorithms or dedicated detection devices, significantly increasing system cost and operational complexity, thus limiting their widespread application in point-of-care testing and low-resource scenarios. Therefore, it is necessary to develop a quantitative one-pot CRISPR-Dx method that requires minimal instrumentation, has a wide dynamic range, and is easy to use in the field. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide reagents and detection methods for semi-quantitative one-pot instantaneous nucleic acid detection.

[0006] The reagents for semi-quantitative one-pot instantaneous nucleic acid detection provided by this invention include RT-RPA reagent, reverse transcription, amplification primers, LbCas12a, crRNA, and ssDNA reporter molecules;

[0007] The LbCas12a has the amino acid sequence shown in SEQ ID NO:17;

[0008] The crRNA targets PAMs such as TTTG, GTTC, TATC, CCCC, GTAG, or ATTG.

[0009] This invention targets one-pot recombinase polymerase amplification (RPA) and CRISPR detection (CRISPR-Dx) reactions. By combining specific LbCas12a with the CRISPR target PAM, a series of one-pot RPA-CRISPR-Dx systems with different limits of detection are constructed. Detection arrays built based on these systems can output semi-quantitative results that require no computation or processing and can be directly read, providing a powerful tool for improving real-time nucleic acid detection quantification capabilities.

[0010] In this invention, the sequence of the crRNA is shown in any one of SEQ ID NO: 11-16.

[0011] The LbCas12a used in this invention exhibits different activities at different PAM sites, leading to variations in detection sensitivity. In this invention, ATTG at the PAM site is used for high-sensitivity detection, while TTTG at the PAM site is used for low-sensitivity detection.

[0012] In this invention, the ssDNA reporter molecule functions as a reporting signal. The 5' end of the ssDNA reporter molecule is modified with a fluorescent group, and the 3' end with a quencher group. When the reporter molecule is intact, the fluorescence emitted by the fluorescent group is effectively quenched by the quencher group. When LbCas12a specifically recognizes and binds to the target nucleic acid sequence under the guidance of crRNA, the paracleavage activity of LbCas12a is activated, thereby non-specifically cleaving the ssDNA reporter molecule in the system. The fluorescent group and the quencher group separate, and the fluorescence signal is released and enhanced. In this invention, the single-stranded oligonucleotide in the ssDNA reporter molecule is a random sequence of 5-10 bp in length. In a specific embodiment, the single-stranded oligonucleotide is 8 bp in length. As a feasible example, the sequence of the single-stranded oligonucleotide is CCCCCCCC. In the ssDNA of this invention, the choice of quencher group and fluorescent group does not significantly affect the detection effect; those skilled in the art can make conventional choices according to actual experimental needs or existing experimental conditions. Specifically, the 5' end modified fluorescent group can be selected from FAM, HEX, TET, VIC, JOE, Cy3, Cy5, or ROX; the 3' end modified quencher group can be selected from BHQ1, BHQ2, BHQ3, TAMRA, DABCYL, MGB, or Eclipse. In some specific embodiments of the present invention, the ssDNA reporter molecule is modified with a FAM fluorescent group at its 5' end and a BHQ1 quencher group at its 3' end.

[0013] The detection method of this invention is based on the RPA reaction, and the RPA reagents involved can be self-made or commercially available. The core components of the RPA reagents are recombinase, polymerase, ssDNA binding protein, deoxynucleoside triphosphates (dNTPs), and buffer solution.

[0014] The reaction principle of the detection reagent of this invention is as follows: The recombinase in the RPA reagent binds to the amplification primers to form a protein-nucleic acid complex. With the cooperation of ssDNA binding proteins, this complex recognizes and binds to the target nucleic acid, using dNTPs for chain extension, achieving isothermal amplification of the target nucleic acid sequence. crRNA specifically recognizes the amplified product site by targeting the PAM sequence. After the complex formed with LbCas12a successfully recognizes and binds to the target sequence, the paracleavage activity of LbCas12a is activated. The activated LbCas12a non-specifically cleaves the ssDNA reporter molecule in the system, causing the 5' fluorescent group to separate from the 3' quencher group, releasing a fluorescent signal. The change in fluorescence intensity of the reaction system can be used to detect the target nucleic acid in the RNA sample.

[0015] When used for DNA detection, the detection system constructed by this reagent includes recombinase, polymerase, ssDNA binding protein, deoxynucleoside triphosphates (dNTPs), primers for amplifying the target nucleic acid, LbCas12a, crRNA, and single-stranded oligonucleotide probes labeled with fluorescent groups and quenchers at both ends. When used for RNA detection, the detection system, in addition to the above components, also includes reverse transcriptase and primers for reverse transcription of the target nucleic acid.

[0016] As a feasibility example, each 100 μL reaction system includes:

[0017] 18 μL RPA reagent, 1 μL reverse transcriptase, 1 μM RPA amplification primers, 50 nM LbCas12a, 50 nM crRNA and 400 nM ssDNA reporter molecule.

[0018] In the detection reagents described in this invention, the principle of primer design is to design one set each of forward (F) and reverse (R) primers on the specific sequence of the nucleic acid to be tested. In the embodiments of this invention, the amplification primers include an upstream primer and a downstream primer, each with an independent length of 20-30 bp. Specifically, a set of primers in the same direction should typically include three lengths: 20, 25, and 30 nt, possessing differentiated sequences and GC content, thereby resulting in differentiated amplification rates. In a specific embodiment, the CG% in the amplification primer sequence is 55%-80%.

[0019] In this invention, the design principle of target PAMs is to identify several PAMs of types such as TTTV, VTTV, TRTV, YYYN, and CCRN on the specific sequence of the nucleic acid to be tested, and then design CRISPR targets downstream of the determined PAMs to create differentiated CRISPR cleavage rates. After the primers and PAMs are designed, they are used as single variables to characterize the impact on the detection signal. Based on the results, different primer pairs and PAMs are combined to construct detection systems with different limits of detection.

[0020] In this embodiment of the invention, to verify the feasibility of the reagent, the PML::RARA fusion transcript was used as the target gene. The amplification primers were primers targeting the bcr1 subtype of the PML::RARA fusion transcript, or primers targeting the bcr3 subtype of the PML::RARA fusion transcript.

[0021] In the primers targeting the bcr1 subtype of the PML::RARA fusion transcript,

[0022] The upstream primer has any one of the nucleic acid sequences shown in SEQ ID NO: 1-3;

[0023] The downstream primer has any one of the nucleic acid sequences shown in SEQ ID NO: 7~10;

[0024] In the primers targeting the bcr3 subtype of the PML::RARA fusion transcript,

[0025] The upstream primer has any one of the nucleic acid sequences shown in SEQ ID NO: 4-6;

[0026] The downstream primer has any one of the nucleic acid sequences shown in SEQ ID NO: 7~10.

[0027] In a specific embodiment, the primers targeting the bcr1 subtype of the PML::RARA fusion transcript have the following sequences: the upstream primer has the nucleic acid sequence shown in SEQ ID NO: 1, and the downstream primer has the nucleic acid sequence shown in SEQ ID NO: 7; or the upstream primer has the nucleic acid sequence shown in SEQ ID NO: 1, and the downstream primer has the nucleic acid sequence shown in SEQ ID NO: 8; or the upstream primer has the nucleic acid sequence shown in SEQ ID NO: 1, and the downstream primer has the nucleic acid sequence shown in SEQ ID NO: 9; or the upstream primer has the nucleic acid sequence shown in SEQ ID NO: 1, and the downstream primer has the nucleic acid sequence shown in SEQ ID NO: 10; or the upstream primer has the nucleic acid sequence shown in SEQ ID NO: 2, and the downstream primer has the nucleic acid sequence shown in SEQ ID NO: 7; or the upstream primer has the nucleic acid sequence shown in SEQ ID NO: 3, and the downstream primer has the nucleic acid sequence shown in SEQ ID NO: 7.

[0028] In a specific embodiment, the primers targeting the bcr3 subtype of the PML::RARA fusion transcript have the following sequences: the upstream primer has the nucleic acid sequence shown in SEQ ID NO: 4, and the downstream primer has the nucleic acid sequence shown in SEQ ID NO: 7; or the upstream primer has the nucleic acid sequence shown in SEQ ID NO: 4, and the downstream primer has the nucleic acid sequence shown in SEQ ID NO: 8; or the upstream primer has the nucleic acid sequence shown in SEQ ID NO: 4, and the downstream primer has the nucleic acid sequence shown in SEQ ID NO: 9; or the upstream primer has the nucleic acid sequence shown in SEQ ID NO: 4, and the downstream primer has the nucleic acid sequence shown in SEQ ID NO: 10; or the upstream primer has the nucleic acid sequence shown in SEQ ID NO: 5, and the downstream primer has the nucleic acid sequence shown in SEQ ID NO: 7; or the upstream primer has the nucleic acid sequence shown in SEQ ID NO: 6, and the downstream primer has the nucleic acid sequence shown in SEQ ID NO: 7.

[0029] By changing primer length and PAM sites, different detection limits can be obtained. In practical testing scenarios, high-sensitivity detection systems are used for early diagnosis, while combinations of different sensitivity detection systems are used for dynamic monitoring. Furthermore, reagents with different sensitivities can improve stability and anti-interference capabilities, reduce instrument dependence and application barriers, and are of significant value in point-of-care testing scenarios. The embodiments described in this application indicate that rapid one-pot nucleic acid detection can be achieved using combinations of different primers and PAMs, with result output time approximately 30-60 minutes. The dynamic range of the detection limit can cover 6-7 orders of magnitude from aM to pM. For the same sample, detection using detection systems containing different detection limits allows for rapid determination of the target nucleic acid content in the sample without relying on any instruments or algorithms. Specifically, the target nucleic acid concentration in the sample is higher than the detection limit of the least sensitive detection system that outputs a positive result, and lower than the detection limit of the most sensitive detection system that outputs a negative result.

[0030] Furthermore, the present invention also provides a one-pot instantaneous nucleic acid detection method, which includes adding the sample to be tested to the reagents as described above, incubating, and then detecting the fluorescence intensity.

[0031] In this invention, the incubation temperature is 35~42℃ and the incubation time is 30~60 min. In a specific embodiment, the incubation temperature is 42℃ and the incubation time is 60 min.

[0032] In some embodiments, the sample to be tested is an RNA sample.

[0033] In this invention, the detection method can be for diagnostic purposes or non-diagnostic purposes. In methods for diagnostic purposes, the target gene being detected is correlated with a disease and / or health condition. In non-diagnostic detection, the target gene is not correlated with a disease and / or health condition, or although the target gene is correlated with a disease and / or health condition, the detection is a qualitative or semi-quantitative analysis of a specific nucleic acid sequence in scientific research.

[0034] This invention targets one-pot recombinase polymerase amplification (RPA) and CRISPR detection (CRISPR-Dx) reactions. Through the redesign of RPA primers and the screening of CRISPR target PAM, and by combining the two, a series of one-pot RPA-CRISPR-Dx systems with different limits of detection are constructed. Detection arrays built based on these systems can output semi-quantitative results that require no computation or processing and can be directly read, providing a powerful tool for improving the quantitative capabilities of real-time nucleic acid detection. Attached Figure Description

[0035] Figure 1 This demonstrates the impact of RPA primer design on the detection signal of one-pot CRISPR-Dx.

[0036] Figure 2 This demonstrates the effect of PAM selection on the detection signal in one-pot CRISPR-Dx.

[0037] Figure 3 Analysis of the limit of detection (LOD) of bcr1 detection system (LOD=10 aM);

[0038] Figure 4 Analysis of the limit of detection (LOD) of bcr3 detection system (LOD=10 aM);

[0039] Figure 5 Analysis of the β-detection limit of the bcr1 detection system (LOD=1 fM);

[0040] Figure 6 Analysis of the β-detection limit of the bcr3 detection system (LOD=1 fM);

[0041] Figure 7 Analysis of the limit of detection (LOD) of γ in the bcr1 detection system (LOD=1 pM);

[0042] Figure 8 Analysis of the limit of detection (LOD) of γ in the bcr3 detection system (LOD=1 pM);

[0043] Figure 9 This demonstrates a semi-quantitative one-pot CRISPR-Dx specificity analysis based on the primer-PAM matrix.

[0044] Figure 10 This indicates the method for reading the detection array results;

[0045] Figure 11 The results of qualitative testing of clinical samples using the bcr1 detection system are shown, where A represents positive samples, B represents negative samples, and C represents sensitivity and specificity analysis.

[0046] Figure 12 The results of qualitative testing of clinical samples using the bcr3 detection system are shown, where A represents positive samples, B represents negative samples, and C represents sensitivity and specificity analysis.

[0047] Figure 13 The results of semi-quantitative detection of clinical samples using the bcr1 detection array (detection systems α, β, γ) are shown, where A is detection system α, B is detection system β, and C is detection system γ.

[0048] Figure 14 The results of semi-quantitative detection of clinical samples using the bcr3 detection array (detection systems α, β, γ) are shown, where A is detection system α, B is detection system β, and C is detection system γ. Detailed Implementation

[0049] This invention provides reagents and detection methods for semi-quantitative one-pot instantaneous nucleic acid detection. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of this invention to realize and apply the technology of this invention.

[0050] Unless otherwise defined in this invention, the scientific and technical terms related to this invention shall have the meanings understood by one of ordinary skill in the art;

[0051] The terms “comprising,” “including,” and “having” are used interchangeably to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed; it should also be understood that the use of “comprising,” “including,” and “having” in this document also provides for schemes that “comprise”.

[0052] The term "and / or" describes the relationship between related objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, A and B exist simultaneously, or B exists alone; where A and B can be singular or plural.

[0053] In this application, "at least one" means one or more, "more" means two or more; "at least one of the following" or similar expressions refer to any combination of these items, including any combination of single items or multiple items;

[0054] The numerical ranges and parameters involved in this invention have been presented as precisely as possible in the specific embodiments; however, any numerical value inevitably contains standard deviations due to individual test methods; therefore, unless otherwise expressly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have a certain reasonable deviation within a certain range, for example: within ±10%, ±5%, ±1% or ±0.5%.

[0055] The embodiments and comparative examples of this invention describe some examples; the embodiments illustrate certain implementations of the invention; however, this does not mean that the effects of the invention can only be achieved in these examples; in fact, good results can be achieved at any concentration of each component between the two endpoint values ​​shown in the embodiments.

[0056] All test materials used in this invention are common commercial products and can be purchased on the market. Among them, the RPA reagent is the DNA isothermal rapid amplification kit (basic type) from Weifang Amp-Future Biotech (Shandong, China).

[0057] The amino acid sequence of LbCas12a involved in the examples is as follows:

[0058]

[0059] The single-stranded oligonucleotide probe sequence labeled with fluorescent groups and quenchers at both ends is as follows:

[0060] FAM-5'-CCCCCCCC-3'-BHQ1 (SEQ ID NO: 18)

[0061] The reagents and methods provided by this invention can be applied to the detection of any target nucleic acid. Specific embodiments involve two detection targets: the bcr1 subtype (L-type) and bcr3 subtype (S-type) of the acute promyelocytic leukemia PML::RARA fusion transcript. RPA primers and crRNA are designed based on the sequences of these two subtypes. However, the detection targets in the specific embodiments are only used to demonstrate the feasibility of the scheme and are not intended to limit the scope of detection targets. The reagents and methods of this invention can also be used for other nucleic acid detection targets, and are not limited to those described in the embodiments of this invention. When detecting other target nucleic acids, primers and crRNA can be redesigned according to the required detection sequence, and screening and combination testing can be performed according to this method.

[0062] The LbCas12a involved in this embodiment of the invention is expressed and purified by the following method: the gene fragment encoding LbCas12a is cloned into a pET-based expression vector containing a C-terminal 6-terminus. His tag. The *E. coli* strain *Rosetta*, transformed with the recombinant plasmid, was cultured to an OD600 of 0.8, then incubated with 0.2 mM isopropyl β-D-1-thiogalactoside (IPTG) at 16 °C for 16 h. Proteins were isolated from cell lysates using Ni-NTA resin and eluted with buffer (50 mM Tris-HCl, 1.5 M NaCl, 5% glycerol, and 600 mM imidazole, pH 8.0).

[0063] The crRNA involved in this invention embodiment was prepared as follows: A crRNA spacer sequence (SEQ ID NO: 11-16) was designed to target a suitable PAM site on the PML::RARA fusion transcript. Oligonucleotides containing the T7 promoter sequence and oligonucleotides containing the spacer sequence were polymerized to obtain a DNA template for in vitro transcription. The template was incubated with T7 RNA polymerase (Vazyme, Nanjing, China) at 37 °C for 2 h for in vitro transcription (IVT). The IVT product was incubated with DNase I (Vazyme, Nanjing, China) at 37 °C for 20 min to remove the template DNA, followed by inactivation of DNase I at 65 °C for 20 min. The obtained crRNA was purified using an RNA purification kit (Genstone Biotech, Beijing, China).

[0064] The nucleic acid amplification method involved in this embodiment of the invention is RPA, and the relevant reagents are from Weifang Amp-Future Biotech (Shandong, China). The RPA amplification primer sequences used are shown in SEQ ID NO: 1-10.

[0065] The reverse transcriptase involved in the embodiments of the present invention is derived from Nanjing Novozymes Biotechnology Co., Ltd. (Vazyme, Nanjing, China).

[0066] The single-stranded oligonucleotide probe labeled with fluorescent groups and quenchers at both ends involved in the embodiments of the present invention is a single-stranded DNA with the sequence 5'-CCCCCCCC-3', wherein the 5' end is labeled with the fluorescent group FAM and the 3' end is labeled with the fluorescent group BHQ1.

[0067] The nucleic acid detection involved in this embodiment of the invention is performed according to the following steps: Following the manufacturer's instructions, one tube of RPA reagent lyophilized powder is resuspended in 29.4 μL of buffer A, and 2.5 μL of buffer B and 14.1 L of DEPC-treated water are added to form an RPA mixture. Each detection reaction system contains 20.5 μL of RPA mixture, 1 μL of reverse transcriptase, 1 μM of RPA amplification primers, 50 nM LbCas12a, 50 nM crRNA, and 400 nM FQ ssDNA reporter molecule (FAM-CCCCCCCC-BHQ). 3 μL of the sample to be tested is added to the obtained reaction system, and then the reaction system is placed in a StepOnePlus real-time quantitative PCR instrument (Thermo Fisher Scientific, USA) and read at 42 °C. It is worth noting that although a real-time quantitative PCR instrument is used to read the detection results in this example, the method of this invention is also compatible with other signal output methods, such as flow chromatography strips, small fluorescence reading devices, etc. Furthermore, although the nucleic acid test in this case targets the PML::RARA fusion transcript (RNA), it can also be used for DNA detection after excluding reverse transcriptase from the detection system.

[0068] The preparation of nucleic acid standard samples involved in this invention embodiment is performed according to the following steps: The target fragment of the bcr1 and bcr3 type PML::RARA fusion gene is synthesized, with a T7 promoter installed upstream. Using the synthesized DNA as a template, the target fragment is transcribed in vitro using T7 RNA polymerase (Vazyme, Nanjing, China). The resulting RNA product is treated with DNase I (Vazyme, Nanjing, China) to remove the DNA template, and then purified using an RNA purification kit (Jianshi Biotechnology, Beijing, China). The purified RNA is serially diluted to specific concentrations for subsequent characterization and validation.

[0069] It should be understood that, in the various embodiments of this application, the sequence numbers of the above processes do not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application. The present invention is further illustrated below with reference to embodiments:

[0070] Example 1: Primers and PAM were used to adjust the detection sensitivity of the one-pot CRISPR-Dx method.

[0071] Experience shows that primer length and GC content significantly affect RPA amplification rate; designing primers with diverse sequences and lengths can lead to variations in RPA amplification rates. In one-pot CRISPR-Dx, detection sensitivity is positively correlated with RPA amplification rate. Therefore, primer design can be used to regulate the sensitivity of one-pot CRISPR-Dx.

[0072] In this embodiment, RPA primers were designed based on the PML::RARA fusion transcript bcr1 subtype (L type) and bcr3 subtype (S type), including two sets of forward primers (LF and SF, corresponding to the L and S subtypes respectively, SEQ ID NO: 1-6) and one set of reverse primers (R, universal for both L and S subtypes, SEQ ID NO: 7-10). Each set of primers has three lengths of 20, 25, and 30 nt and different GC contents (Table 1).

[0073] Table 1 Primers for PML::RARA fusion transcript detection

[0074]

[0075] Based on the different primers used, the subjects were divided into 6 test groups (denoted as F1R1, F1R2, F1R3, F1R4, F2R1, and F3R1, respectively) to detect BCR1 and BCR3.

[0076] The primers involved in the test group F1R1 included L-F1 + S-F1 + R1.

[0077] The primers involved in the test group F1R2 included L-F1 + S-F1 + R2.

[0078] The primers involved in the test group F1R3 included L-F1 + S-F1 + R3.

[0079] The primers involved in the test group F1R4 included L-F1 + S-F1 + R4.

[0080] The primers involved in the test group F2R1 included L-F2 + S-F2 + R1.

[0081] The primers involved in the test group F3R1 included L-F3 + S-F3 + R1.

[0082] Using the same crRNA (SEQ ID NO: 16), the signal intensity of different RPA primers in the corresponding 1 fM standard nucleic acid one-pot detection was examined.

[0083] The results showed that for the three groups of RPA primers (LF, SF, and R) with different annealing positions, primers with different lengths, sequences, and GC contents within each group could produce detection signals of different intensities, indicating that the selection of RPA primers can be used as a means to regulate the activity of one-pot CRISPR-Dx. Figure 1 ).

[0084] Cas12a can recognize multiple PAM sites and exhibits different activities at different PAM sites. The differences in Cas12a cleavage activity caused by targeting different PAM sites will affect the sensitivity of one-pot CRISPR-Dx, thereby achieving regulation. In this embodiment, the crRNA is designed based on the shared sequence of the PML::RARA fusion transcripts bcr1 (L-type) and bcr3 (S-type) to target sites with different PAM sequences. Empirically, available PAMs include, but are not limited to, VTTV, TTTV, TRTV, YYYN, CCRN, etc., and the sensitivity of these PAMs in one-pot CRISPR-Dx generally decreases in the above order. The PAMs involved in this embodiment include TTTG, GTTC, TATC, CCCC, GTAG, and ATTG (Table 2, SEQ ID NO: 11-16).

[0085] Table 2. Detection of crRNA from PML::RARA fusion transcripts

[0086]

[0087] Using the same RPA primer pair (SEQ ID NO: 1, 4, 7), the signal intensity of different crRNAs targeting different PAMs in the corresponding 1 fM standard nucleic acid one-pot assay was investigated. The results showed that crRNAs targeting different PAMs could produce detection signals of varying intensities, indicating that PAM selection can serve as a means of regulating the activity of one-pot CRISPR-Dx assays. Figure 2 The strongest detection signal was found in the test group using a targeted PAM with a VTTV structure, where V represents A, G, or C.

[0088] Example 2: Primer-PAM matrix for semi-quantitative one-pot CRISPR-Dx array construction

[0089] Semi-quantitative results are output based on a CRISPR-Dx array. The array contains three (or more) one-pot CRISPR-Dx systems with different limits of detection. The detection system includes different combinations of primers and PAM, which work together to adjust the sensitivity of the CRISPR-Dx detection system, thereby determining the limit of detection at the desired level.

[0090] In this embodiment, a detection system composed of different primers and PAM was used to detect the graded dilution of PML::RARA fusion transcript standard RNA (Table 3).

[0091] Table 3 APL Detection System

[0092]

[0093] Experimental results show that using high-performance primers of 30 nt in length combined with ATTG-type PAM sites can achieve high amplification efficiency, while the Cas12a cleavage activity is relatively weakened, bringing the amplification and cleavage processes to a near-equilibrium state, thus obtaining a detection limit as low as approximately 10 aM. This detection system is called α( Figure 3-4 When using primers with slightly lower performance and paired with classic PAM sites, the amplification efficiency can still be maintained, but the enhanced Cas12a cleavage activity shifts the reaction system towards the cleavage process, increasing the detection limit to approximately 1 fM. This detection system is called β( Figure 5-6 ).

[0094] When using primers with lower performance and combining them with classic PAM sites, the amplification efficiency decreased significantly while the cleavage activity increased. The reaction system became more biased towards the cleavage process, and the detection limit increased to approximately 1 pM. This detection system is called γ( Figure 7-8 The above detection system was used to detect nucleic acids from Pseudomonas aeruginosa, Helicobacter pylori, monkeypox virus, and Severe Acute Respiratory Syndrome Coronavirus 2. All results were negative, indicating that the detection method has high specificity. Figure 9 ).

[0095] When performing qualitative detection on a target sample, only the detection system with the highest sensitivity is required. When performing semi-quantitative detection on a target sample, detection systems with different sensitivities must be used simultaneously. The range of Mycoplasma pneumoniae nucleic acid concentration in the sample is determined based on the number of detection systems that output positive results. In this embodiment, all three systems output positive results, indicating that the target nucleic acid content exceeds 1 pM; two systems output positive results and one system outputs a negative result, indicating that the target nucleic acid content is between 1 fM and 1 pM; one system outputs a positive result and two systems output negative results, indicating that the target nucleic acid content is between 10 aM and 1 fM; all three systems output negative results, indicating that the target nucleic acid content is below 10 aM (…). Figure 10 ).

[0096] Example 3: Semi-quantitative one-pot CRISPR-Dx array for APL clinical sample diagnosis and monitoring

[0097] In this embodiment, a primer-PAM matrix-mediated one-pot CRISPR-Dx array was used to detect clinically derived samples to verify the qualitative and semi-quantitative detection performance of the method in real samples.

[0098] Bone marrow mononuclear cell samples and peripheral blood samples were collected from clinical sources. The bone marrow mononuclear cell samples included:

[0099] 1) 28 PML-RARA transcript positive samples were confirmed by RT-qPCR, of which 21 were bcr1 subtype (L type) and 7 were bcr3 subtype (S type).

[0100] 2) A total of 18 samples were collected, including other leukemia subtypes without PML-RARA transcripts and negative control samples without PML-RARA transcripts.

[0101] A total of 75 peripheral blood samples were collected, all from subjects negative for the PML-RARA transcript. To simulate a clinical testing scenario, known concentrations of PML-RARA RNA standards were added to the peripheral blood samples to prepare simulated samples with different target concentrations, including 30 samples of the bcr1 subtype and 44 samples of the bcr3 subtype. All samples were anonymized before testing and did not involve any personally identifiable information.

[0102] Bone marrow mononuclear cell samples were lysed and RNA extracted using Trizol reagent; peripheral blood samples were lysed and RNA extracted using Trizol LS reagent. The obtained RNA was purified using a column purification kit. The purified RNA samples were detected using both the reference method (RT-qPCR) and the primer-PAM matrix-mediated one-pot CRISPR-Dx array described in this invention for result comparison. The CRISPR-Dx array consists of multiple one-pot detection systems with different preset detection limits. The detection system with the lowest detection limit (α system) is used for qualitative determination of the target nucleic acid, and all detection systems (α, β, γ systems) are used together for semi-quantitative analysis.

[0103] For each subtype, a total of 51 positive samples were used for qualitative detection using the detection system with the lowest detection limit (α system). 50 samples were positive for bcr1, and 51 samples were positive for bcr3. Of the 18 negative samples, the detection system for bcr1 yielded negative results in 17 cases, with one false positive; the detection system for bcr3 was negative for all 18 samples. These results indicate that primer-PAM matrix-mediated CRISPR-Dx has a sensitivity of 98% and a specificity of 94.4% for qualitative detection of bcr1; and a sensitivity and specificity of 100% for bcr3. Figure 11-12 ).

[0104] Further, 23 bcr1 samples and 22 bcr3 samples were randomly selected from spiked peripheral blood samples, and semi-quantitative detection was performed using a one-pot CRISPR-Dx array (containing α, β, and γ systems) mediated by the primer-PAM matrix. The results showed that the semi-quantitative interpretation of the array output was consistent with the target nucleic acid levels reflected by the reference method RT-qPCR. Specifically, in the bcr1 samples, 6 samples had target nucleic acid concentrations in the 10 aM–1 fM range, 8 samples were in the 1 fM–1 pM range, and 7 samples were above 1 pM; in the bcr3 samples, 9, 5, and 8 samples, respectively, fell within the corresponding ranges. Figure 13-14 ).

[0105] The combined detection results of bone marrow mononuclear cell samples and spiked peripheral blood samples show that the primer-PAM matrix-mediated one-pot CRISPR-Dx detection method of the present invention has both reliable qualitative detection capability and semi-quantitative interpretation capability in actual samples, and can be used for the detection of target nucleic acids and the diagnosis and monitoring of related diseases.

[0106] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Reagents for semi-quantitative one-pot instantaneous nucleic acid detection, including RPA reagent, reverse transcriptase, amplification primers, LbCas12a, crRNA, and ssDNA reporter molecules; in, The LbCas12a has the amino acid sequence shown in SEQ ID NO:17; The crRNA targets PAMs such as TTTG, GTTC, TATC, CCCC, GTAG, or ATTG.

2. The reagent according to claim 1, characterized in that, The sequence of the crRNA is shown in any one of SEQ ID NO: 11-16.

3. The reagent according to claim 1, characterized in that, In the ssDNA reporter molecule, the single-stranded oligonucleotides are random sequences with a length of 5-10 bp. The fluorescent group modified at its 5' end is selected from FAM, HEX, TET, VIC, JOE, Cy3, Cy5, ROX or TAMRA; The quenching group modified at its 3' end is selected from BHQ1, BHQ2, BHQ3, TAMRA, DABCYL, MGB, or Eclipse.

4. The reagent according to claim 1, characterized in that, The RPA reagent includes recombinase, ssDNA binding protein, polymerase, dNTPs, and buffer.

5. The reagent according to claim 1 or 4, characterized in that, Each reaction system includes: 18 μL RPA reagent, 1 μL reverse transcriptase, 1 μM RPA amplification primers, 50 nM LbCas12a, 50 nM crRNA and 400 nM ssDNA reporter molecule.

6. The reagent according to any one of claims 1 to 5, characterized in that, The amplification primers include an upstream primer and a downstream primer, and the upstream primer and the downstream primer are of independent lengths of 20~30bp; The CG% in the amplification primer sequence is 55%~80%.

7. The reagent according to claim 6, characterized in that, The amplification primers are primers targeting the bcr1 subtype of the PML::RARA fusion transcript, or primers targeting the bcr3 subtype of the PML::RARA fusion transcript. In the primers targeting the bcr1 subtype of the PML::RARA fusion transcript, The upstream primer has any one of the nucleic acid sequences shown in SEQ ID NO: 1-3; The downstream primer has any one of the nucleic acid sequences shown in SEQ ID NO: 7~10; In the primers targeting the bcr3 subtype of the PML::RARA fusion transcript, The upstream primer has any one of the nucleic acid sequences shown in SEQ ID NO: 4-6; The downstream primer has any one of the nucleic acid sequences shown in SEQ ID NO: 7~10.

8. A method for one-pot instantaneous nucleic acid detection, comprising adding the sample to be tested to the reagent described in any one of claims 1 to 7, incubating, and then detecting the fluorescence intensity.

9. The method according to claim 8, characterized in that, The incubation temperature is 35~42 ℃, and the time is 30~60 min.

10. The method according to claim 8 or 9, characterized in that, The sample to be tested is an RNA sample.

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